Systems and methods involving data patterns such as spectral biomarkers

ABSTRACT

The present invention is generally related to the separation, fractionation, and/or characterization of molecules and/or biomolecules in one or more mixtures. After fractionation, different phases of a partitioning system can be analyzed via an analytical technique such as spectral analysis, chromatography, or the like, to produce a spectrum or other symbolic representation of the species after fractionation, and the spectra of the various fractions/phases compared to define a comparative spectrum as a marker or otherwise providing information about the sample, including such information that is independent of the original level of abundance of the molecules in the mixture. Comparative spectra of various samples can be compared to each other and/or to controls or reference spectra and/or comparative spectra to determine a variety of information. In some embodiments, the methods can be used for discovering and/or identifying patterns in a mixture of species and/or corresponding patterns of species in a second mixture, where each mixture of species originates from biological systems with different physiological conditions as markers associated with specific diagnostics, and can be used for screening for such markers once discovered and identified during diagnostics screening.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/751,715, filed Dec. 19, 2005, entitled “Systemsand Methods Involving Spectral Biomarkers,” by Chait, et al.,incorporated herein by reference.

FIELD OF INVENTION

The present invention is generally related to the characterization ofphysical samples from the analysis of at least one component or speciesof the sample. In some specific embodiments, the invention relates tofractionation of species within a samples and comparative analysis ofdifferent fractions to define comparative data, which can be compared toother, similar data to provide information regarding the sample and/or aspecies within the sample.

BACKGROUND

Many diseases and/or other pathological processes or conditions arecaused by dysfunction and/or disregulation of certain proteins. Thesedisease-related proteins may have their structures altered, relative totheir “normal” or “wild-type” counterparts, and/or may be expressed inlarger (up-regulated expression) or smaller (down-regulated expression)quantities in a given disease state, relative to “normal” physiologicalconditions. In some cases, proteins having altered structure and/orfunction may be used as markers associated with a particular human oranimal disease, for instance, as a diagnostic for earlier detection ofthe disease, or the like. In many cases, the particular protein(s) ofrelevance to a given pathological process of a disease or othercondition are unknown. Identification of such protein(s) would be usefulfor development of new diagnostic tests, or the like.

A general approach to the identification and characterization of proteinmarkers is based on the analysis of protein compositions of samples ofbiological material (biological fluids, such as blood, serum, plasma,cerebrospinal fluid,.tissues, cells, etc.) using high resolutionseparation techniques. For instance, proteins isolated from control andexperimental samples or populations can be subjected to proteolyticcleavage, and their cleavage products identified using liquidchromatography (LC) coupled with tandem mass spectrometry (LC-MS-MS).Many protein separation techniques are based on multi-dimensionalseparation of proteins from a sample, typically by two-dimensional gelelectrophoresis (2-DE) or two-dimensional high-performance liquidchromatography (2D-HPLC). The two-dimensional (2-D) protein maps forpathological samples may be obtained and compared with those forreference samples; positions of proteins observed as “spots” on (2-DE)maps or as “peaks” on 2D-HPLC maps can be compared, and those that arepresent (or absent) in the maps obtained from pathological samples butabsent (or present) in the maps obtained from the reference samples maybe judged as being likely to correspond to pathologically relevantproteins. Additionally, quantities of proteins estimated as intensitiesof the spots (or peaks) may be evaluated and compared between thepathological and reference samples. Those that are significantlydifferent may be considered as pathologically relevant in some cases.

It has also been recently established that a pattern of thepresence/absence and/or the relative quantities of multiple proteins (a“signature”) may also be of diagnostic relevance, where the proteinsjudged to be of interest are identified by peptide mapping and/or massspectrometry. Mathematical or statistical techniques, such as patternrecognition techniques, can be used to analyze the pattern produced bythese experimental techniques and produce a diagnostic classification.However, this approach is often highly inefficient, for example, due tothe inherent necessity of analyzing all of the proteins in a givensample, whereas only a small portion of the proteins may have anypathological relevance.

Several different methods for reducing the analytical complexity ofprotein mixtures have been developed. These methods are typically basedon fractionation of the original mixture prior to 2D analysis by gelelectrophoresis or 2D-HPLC. One such method is the separation ofproteins by the technique of free-flow electrophoresis. However, thistechnique, while fractionating the original protein mixture, may resultin multiple 2D analysis of simplified fractions, i.e. while reducing thecomplexity of analysis and improving resolution, it inherently greatlyincreases the number of samples where further analysis is required.

Another method is fractionation based on the affinity of proteins todifferent natural ligands and/or pharmacological compounds; however,this approach, while allowing separation of proteins according toprotein functions, may result in a large increase in the number ofsamples for further analysis, and often requires additional knowledge orpresumption concerning the differences between the samples.

One disadvantage of most fractionation techniques is that they generallycannot preserve protein-protein or protein-ligand interactions.Differences among biological interactions are often important forelucidating and detecting changes among samples. Additionally, most ofthe fractionation techniques rely on separation due to a fixed physicalattribute, such as molecular size or net charge. While these attributesmay be very important for distinguishing among biomolecules in a complexmixture, they generally do not cover all of the potential differencesbetween biomolecules representing, e.g., normal vs. disease states,differences in configuration etc. Another important disadvantage ofpresent fractionation techniques is related to their inability toseparate mixtures based on differences between structural changes in,e.g., glycosylation patterns and/or conformational changes. Thesechanges are often important for identifying proteins that eitherparticipate in and/or are the result of a disease state. For example, ifa protein is misfolded as a result of genetic mutation, the net chargeand size of the protein may not vary significantly, and moreimportantly, the protein's expression level might be the same for theunderlying normal vs. disease states. Finally, natural geneticvariability among individuals can significantly contribute to a verylarge scatter in the expression levels (concentrations) of biomoleculesin a biological sample. This variability generally necessitates use ofstatistically large number of samples to robustly detect differencesinnate to a particular pathological condition, rather than to geneticvariability. Natural genetic variability is often a significanthindrance in implementing protein marker based diagnostics due toreduction of the sensitivity and/or specificity of the diagnostic test.

While significant advances in the field of molecular and/or samplecharacterization have been made, improvements are therefore needed toadd specificity, versatility, convenience, and/or improve efficiency.

SUMMARY OF THE INVENTION

The present invention is generally related to the separation,fractionation, and/or characterization of a mixture of molecules and/orbiomolecules or other species. For example, in some embodiments, thepresent invention provides systems and methods for the analysis andcharacterization of mixtures of biomolecules, complexes comprisingbiomolecules, molecules which interact with biomolecules, and/oranalogous species thereof. For example, differences in overall patternsof analyses of mixtures of biomolecules may indicate protein markers ofa disease and/or a physiological state of a living organism.

The subject matter of this application may involve, in some cases,interrelated products, alternative solutions to a particular problem,and/or a plurality of different uses of a single system or article.

One aspect of the invention is directed to a method of determining acharacteristic of a plurality of species. The method, according to oneset of embodiments, includes acts of exposing a plurality of species toat least first and second interacting components defining at least afirst phase and a second phase, respectively, of a first system thatincludes at least two phases, obtaining a first spectral data patterncomprising cumulative spectral information from a plurality of speciesof the first phase of the system after exposure, obtaining a secondspectral data pattern comprising cumulative spectral information from aplurality of species of the second phase of the system after exposureand/or cumulative spectral information from a plurality of the pluralityof species prior to exposure to the system, and deriving comparativespectral information from comparison of at least a portion of the firstspectral data pattern with at least a portion of the second spectraldata pattern, to determine a characteristic of a plurality of species.

In one embodiment, the invention involves developing and using methodsfor utilizing the effects of differences in relative measures ofinteraction of species with different phases of multi-phase systems,e.g. fractionation or separation, for example via multi-phasepartitioning, of two, three, or more mixtures, which may reflectdifferences between the mixtures related to the structural and/orfunctional characteristics of a mixture of molecules and/or moleculeswhich interact with such molecules. These techniques can be used, forinstance, to identify unique patterns of such markers using massspectrometry or other analyses in samples, and/or to use such patternsof markers for diagnostics and other related applications.

In one aspect, the invention is a method of determining a characteristicof a plurality of species. In one set of embodiments, the methodincludes acts of exposing a plurality of species to, and causing theplurality of species to interact differently relative to each other uponsaid exposure to, at least first and second interacting componentsdefining at least a first phase and a second phase, respectively, of afirst system that includes at least two phases; obtaining a firstspectral data pattern comprising cumulative spectral information from aplurality of species of the first phase of the system after exposure,which spectral data pattern is representative of the effect of such ofthe relative measures of interaction of the species with the differentphases; obtaining a second spectral data pattern comprising cumulativespectral information from a plurality of species of the second phase ofthe system after exposure, and/or cumulative spectral information from aplurality of the original plurality of species; and deriving comparativespectral information from at least a portion of the first spectral datapattern and the second spectral data pattern to determine acharacteristic of a plurality of species.

In another set of embodiments, the method includes acts of partitioninga plurality of species between a first phase and a second phase of apartitioning system that includes at least two phases; obtaining a firstspectral data pattern comprising cumulative spectral information from aplurality of species of the first phase of the system afterpartitioning; obtaining a second spectral data pattern comprisingcumulative spectral information from a plurality of species of thesecond phase of the system after partitioning, and/or cumulativespectral information from a plurality of the original plurality ofspecies; and deriving comparative spectral information from at least aportion of the first spectral data pattern and the second spectral datapattern to determine a characteristic of a plurality of species.

In another aspect, the invention involves determining a physiologicalcondition of a biological system. In one embodiment, a method for doingso involves determining a comparative pattern from a mixture of speciesof a sample from a biological system, where the comparative pattern isderived from patterns of data obtained from analysis of at least firstand second interacting components defining at least a first phase and asecond phase, respectively, of a first partitioning system. From theprocess of determining the comparative pattern between the mixture ofspecies and the first and second interacting components of the firstpartitioning system, the physiological condition of the biologicalsystem can be determined.

In another embodiment, the method involves determining a physiologicalcondition of a biological system by determining a difference between thecomparative pattern described herein that was obtained from a biologicalsystem and a corresponding comparative pattern representative of areference condition of the biological system, without knowledge of thechemical or biological identity of the individual species in the mixtureof species that result in such patterns.

In another embodiment, a method involves determining a physiologicalcondition of a biological system by determining a difference and/orsimilarity between a first property and/or value of a propertyassociated with a comparative pattern obtained from the biologicalsystem and the comparative patterns obtained from at least one samplewith at least one reference condition.

In yet another embodiment, the method involves determining thephysiological condition of a biological system by determining thedifference and/or similarity between mathematically or statisticallyprocessed analysis patterns obtained from the biological system, andsimilarly mathematically or statistically processed comparative patternsof relative measures of interaction obtained from at least one samplewith at least one reference condition.

In another aspect, the invention relates to a method of identifying oneor more tools for physiological analysis. In one embodiment, the methodinvolves determining a comparative pattern between the data patternsobtained from analyses of species comprising a first mixture of speciesand at least first and second interacting components defining at least afirst phase and a second phase, respectively, of a first partitioningsystem. A comparative pattern also determined likewise between thespecies comprising a second mixture of species, corresponding to thespecies of the first mixture of species, and the first system. Adifference is determined in the comparative pattern of the species ofthe first mixture, versus the comparative pattern of the species of thesecond mixture, with the first system. Based upon this difference, afirst system is selected as a tool for determining a physiologicalcondition of a biological system. Alternatively, or in addition, thecomparative pattern of the species comprising the first mixture and thecomparative pattern of the species comprising the second mixture areselected for determining a physiological condition of a biologicalsystem.

The invention, in still another aspect, is directed to a method ofdetermining at least one characteristic of a plurality of species. Themethod, according to one set of embodiments, includes acts of exposing aplurality of species to an aqueous partitioning system including atleast first and second phases; obtaining, using mass spectroscopy, afirst spectral data pattern comprising cumulative spectral informationfrom a first sample of one or more species associated with the firstphase of the aqueous partitioning system; obtaining, using massspectroscopy, a second spectral data pattern from one or more of thefollowing: (1) a second sample of one or more species associated withthe second phase of the aqueous partitioning system, or (2) a portion ofthe plurality of species prior to the exposing step; and comparing atleast a portion of the first spectral data pattern with at least aportion of the second spectral data pattern to determine at least onecharacteristic of a plurality of species.

The method, in another set of embodiments, includes acts of exposing aplurality of species to at least first and second interacting componentsto at least partially separate the plurality of species; treating afirst sample of the at least partially separated plurality of species,using mass spectroscopy, to produce a first spectral data pattern;treating one or more of the following, using mass spectroscopy, toproduce a second spectral data pattern: (1) a second sample of the atleast partially separated plurality of species that is not identical tothe first sample, or (2) a portion of the plurality of species prior tothe exposing step; and comparing at least a portion of the firstspectral data pattern with at least a portion of the second spectraldata pattern to determine at least one characteristic of a plurality ofspecies.

In still another set of embodiments, the methods exposing a plurality ofspecies to an aqueous partitioning system including at least first andsecond phases; obtaining a first data pattern comprising cumulativeinformation from a first sample of one or more species associated withthe first phase of the aqueous partitioning system; obtaining a seconddata pattern comprising cumulative information by treating one or moreof the following: (1) a second sample of one or more species associatedwith the second phase of the aqueous partitioning system, or (2) aportion of the plurality of species prior to the exposing step; andcomparing at least a portion of the first data pattern with at least aportion of the second data pattern to determine at least onecharacteristic of a plurality of species.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more documents incorporated by reference include conflicting and/orinconsistent disclosure with respect to each other, then the documenthaving the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figure, which isschematic and not intended to be drawn to scale. In the figure, eachidentical or nearly identical component illustrated is typicallyrepresented by a single numeral. For purposes of clarity, not everycomponent is labeled, nor is every component of each embodiment of theinvention shown where illustration is not necessary to allow those ofordinary skill in the art to understand the invention. In the figures:

FIG. 1 is a schematic block diagram of a process for conducting thedetermining and using the patterns of the relative measures ofinteraction according to one embodiment of the present invention;

FIG. 2 is a schematic block diagram of comparison of comparative massspectral information from partitioning systems providing information tothe user, in accordance with the invention;

FIG. 3 is a comparative spectrum, derived from comparison of 2D-HPLCchromatograms of aliquots from different phases of a two-phasepartitioning system after fractionation of a healthy (control) plasmasample;

FIG. 4 is a comparative spectrum derived as that of FIG. 3, but from apatient previously diagnosed with posttraumatic stress disorder;

FIG. 5 shows a comparison of spectral K vectors for two samples,according to one embodiment of the invention;

FIG. 6 illustrates certain biomarkers useful for distinguishing betweenearly and late stage ovarian cancer, according to another embodiment ofthe invention; and

FIG. 7 shows data illustrating differences between normal and cancersubjects for a relative measure of interaction, in yet anotherembodiment of the invention.

DETAILED DESCRIPTION

The present invention is generally related to the interaction of aplurality of molecules or other species with media that can causeseparation of at least some of the molecules or species from each other,and treatment of at least one portion of molecules/species resultingfrom separation to define a pattern. The pattern can be compared to apattern from another portion of molecules/species resulting fromseparation, and/or from a pattern obtained from the original pluralityof molecules or other species prior to separation, resulting incharacterization of the plurality of molecules and/or biomolecules orother species, and/or characterization of a condition associated with anentity associated with the molecules/species with or without anyspecific characterization of any molecules or biomolecules, etc. Theseparation media can include multi-phase partitioning systems or otherseparation materials discussed below. The mixture may be partially orfully separated. In some embodiments, the present invention providessystems and methods for the detection, identification, and/orcharacterization of differences between properties or behavior ofcorresponding patterns of data, for example, measures of interaction ofspecies in the mixture. Any technique may be used to generate thepattern, for example spectral analysis techniques such as massspectroscopy, NMR spectroscopy, UV and/or visible spectroscopy, etc. Theplurality of molecules may include biomolecules and/or molecules able tointeract with biomolecules. The pattern may be produced using samples ofthe mixture (before or after separation), depending on the comparison tobe achieved.

One aspect of the invention involves use of spectral data (as a pattern)obtained from, e.g., a mass spectrometer, obtained from a sample or afraction of a sample, for example a sample of biological origin, wherethe sample is first allowed to interact with an interacting system. Theinteracting system interacts preferentially with some of the species inthe sample to result in at least one (and sometimes more than one)fraction of the sample that is different from the original sample. Thespectral data is then compared with either another spectral dataobtained from a second fraction of the same sample which was allowed tointeract with the same interacting system, or from the spectral data ofthe sample itself.

However, it should be understood that the invention is not limited tothe use of only spectral data. In general, any method of treatment of asample that can be used to produce a data pattern can be used. Forexample, a portion of a sample or fraction of a sample may be treated toproduce a data pattern using any suitable technique, for example, NMRspectroscopy, UV and/or visible spectroscopy, IR spectroscopy, Ramanspectroscopy, fluorescence spectroscopy, mass spectroscopy,chromatography (e.g., liquid chromatography, HPLC, chromatographicelution profile analyses, etc.), GPC, ELIZA, scintillation counting,etc. In some cases, the data itself may be treated to produce a datapattern, e.g., by mathematical processing, data transformation, datasmoothing, noise filters, etc. Accordingly, it should be noted that whendiscussions herein refer to “spectral data” or “comparative spectra,”this is by way of example only, and other data patterns or comparativedata patterns described herein may also be used, in other embodiments ofthe invention.

In one aspect, the invention involves partitioning and in some cases,following partitioning, the partitioning components can be subjected tospectral analysis such as mass spectral analysis, or the like, or othersuitable methods for producing a data pattern, without the need toanalyze spectra of different components and without the need, in certainembodiments, to attempt to identify characteristics of those components(although this can be done in some embodiments). Spectral data or otherdata patterns of different components or phases, and/or species withinthem, can be compared in some embodiments to define a comparativespectrum or pattern which, in and of itself, can be a marker associatedwith the mixture of species and/or can reveal information about themixture of species. This information can include information about thecondition of an entity from which the species was derived, for example,a medical condition and/or a physiological condition of a patient whenthe comparative spectrum or pattern is compared to a referencecomparative spectrum or pattern defining a control. The comparativespectrum or pattern can be derived in some cases from the spectra orother data pattern of the components or phases using one or moremathematical operations, such as subtraction, division, multiplicationor other transformation. In one embodiment, a comparative spectrum orpattern is obtained by a point-by-point division of the data pattern ofthe components or phases, since a division is also a normalizationoperation. A normalization operation in the present context refers tocancellation or removal of the absolute level of the data patternattributable to the underlying protein, protein fragment, or peptidethat resulted in a specific peak or other specific information in thetwo or more patterns being compared. Thus, a comparative spectrum orpattern that is obtained by dividing the data patterns of components orphases using point-by-point division only exhibits relative changesbetween the affinity of the underlying proteins to each of the phasesand not to their absolute quantities in the original sample.

Other signal processing techniques and transformations can be used topre- or post-process the data pattern before or after construction ofthe comparative pattern. Such techniques include, but are not limitedto, data smoothing, noise filters, interpolation, etc. of the spectra,with or without transformations such as Fourier or wavelet transforms,etc., and with or without the use of digital or other filters.

For example, a control can be established by withdrawing a sample from areference entity, such as a blood sample from a healthy patient (anyother sample such as urine, plasma, and those known in the art can beused and/or further treated prior to use according to standardtechniques). The sample can be subjected to fractionation in amulti-phase partitioning system (or any other technique disclosedherein). Where a two-phase partitioning system is used, at least aportion or portions of the first and second phases (or, optionally, morephases, if more phases exist) can be subjected to spectral analysis (orother data patterning technique disclosed herein) and their spectra (orother data pattern) compared to create a comparative spectrum.Alternatively, or in addition, the original sample prior to partitioningcan be subjected to spectral analysis and its spectral data compared toone or more items of spectral data from the first and/or second phasesto create a comparative spectrum. In some of these arrangements, anynumber of spectra of the mixture prior to partitioning, and any numberof phases after the partitioning can be obtained. In some cases, atleast two spectral data are compared to obtain information defining thecontrol (optionally in combination with other information such astemperature, blood pressure, or the like). The comparative spectrum can,optionally, be stored for later use, for example stored as a paperprintout of the spectral comparison, electronically stored in a computeror on any other known storage medium.

Where there is a question as to whether a particular entity exhibits aparticular condition, for example, whether a patient has a particularphysiological condition, then a sample (e.g., an analogous, similar, oridentical sample as that of a control) can be withdrawn from the entityand subjected to systems that can at least partially separate the sample(e.g., a partitioning system). The data may be analyzed using the datafrom the partitioning phase or phases and/or the data pattern of theoriginal, pre-partitioning mixture, and then compared (e.g., by forminga comparative spectrum). The comparative spectrum of the unknown can becompared to the comparative spectrum of a control to determine whetherthe sample substantially deviates from the control, or is essentiallythe same as the control, which can give indication as to whether thesample indicates disease in a patient or not. The control can be, ofcourse, that of a healthy patient or that of a patient having any of anumber of physiological dysfunctions or diseases. Alternatively, two ormore controls can be used defining a healthy state and/or any of anumber of dysfunctional states and the comparative spectrum or patternfrom partitioning of the unknown sample can be compared to any or all ofthese controls to determine a state of the entity from which the samplewas taken. In practice, multiple comparative spectra or patternsoriginating from different entities that exhibit the same dysfunctionalstate can be averaged to form a composite comparative spectrum orpattern.

In some embodiments, the methods can be used for discovering and/oridentifying patterns in a mixture of species and/or correspondingpatterns of species in a second mixture, where each mixture of speciesoriginates from biological systems with different physiologicalconditions as markers associated with specific diagnostics, and can beused for screening for such markers once discovered and identifiedduring diagnostics screening.

The following documents are incorporated herein by reference in theirentirety: U.S. Pat. No. 6,136,960, issued Oct. 24, 2000, entitled“Method for Evaluation of the Ratio of Amounts of Biomolecules or TheirSub-populations in a Mixture,” by Chait et al.; PCT Publication No. WO03/042694, published May 22, 2003 entitled “Characterization ofMolecules,” by A. Chait, et al.; U.S. Patent Application Ser. No.60/478,645, filed Jun. 13, 2003, entitled “Systems and Methods forCharacterization of Molecules,” by A. Chait, et al.; U.S. PatentApplication Ser. No. 60/561,945, filed Apr. 14, 2004, entitled “Systemsand Methods for Characterization of Molecules” by Chait, et al.;International Patent Application No. PCT/US04/19343, filed Jun. 14,2004, entitled “Systems and Methods for Characterization of Molecules,”by A. Chait, et al.; U.S. Patent Application Ser. No. 60/634,586 filedDec. 9, 2004, entitled “Spectral and Other Analysis of PartitionedSamples,” by A. Chait, et al.; and U.S. Patent Application Ser. No.60/751,715, filed Dec. 19, 2005, entitled “Systems and Methods InvolvingSpectral Biomarkers,” by A. Chait, et al.

Definitions

As used in the specification and the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a biomolecule” caninclude mixtures of biomolecules, and the like.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

“Analyte,” “analyte molecule,” or “analyte species” refers to amolecule, for example, a macromolecule, such as a polynucleotide orpolypeptide, whose presence, amount, and/or identity are to bedetermined.

“Antibody,” as used herein, means a polyclonal or monoclonal antibody.Further, the term “antibody” includes, but is not limited to, intactimmunoglobulin molecules, chimeric immunoglobulin molecules, or Fab orF(ab′)₂ fragments. Such antibodies and antibody fragments can beproduced by techniques well known in the art, which include, forexample, those described in Harlow and Lane (Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)),Kohler et al. (Nature 256: 495-97 (1975)), each incorporated herein byreference. Correspondingly, antibodies, as defined herein, also includesingle chain antibodies (ScFv), which may comprise linked V_(H) andV_(L) domains and which may retain the conformation and the specificbinding activity of the native idiotype of the antibody. Such singlechain antibodies are well known in the art and can be produced bystandard methods. See, e.g., Alvarez et al., Hum. Gene Ther. 8: 229-242(1997)). The antibodies of the present invention can be of any isotype,for example, IgG, IgA, IgD, IgE, or IgM.

“Aqueous,” as used herein, refers to the characteristic properties of asolvent/solute system wherein the solvating substance has apredominantly hydrophilic character. Examples of aqueous solvent/solutesystems include those where water, or compositions containing water, arethe predominant solvent. In one embodiment, an aqueous material ismiscible in water, and does not form a separate, identifiable phaseapart from water after being left undisturbed for a day under ambientconditions (e.g., at 1 atm and room temperature, about 25° C. (Note thatwater is miscible in itself.)

A “partitioning system,” as used herein, refers to any material havingat least two phases, sections, areas, components, or the like, at leasttwo of which can interact differently with at least one species to whichthey are exposed. For example, a partitioning system can includedifferent areas of a solid surface, which can interact differently witha particular molecule exposed to the different sections (e.g., as inliquid chromatography or HPLC, etc.), a multi-phase system such as amulti-phase liquid system, e.g., an aqueous/non-aqueous system or anaqueous multi-phase system (defined below) to which one or more speciescan be exposed and optionally dissolved, at least some of which speciescan interact differently with different phases. For example, aparticular species may have a greater affinity for one phase rather thananother phase to the extent that a multi-phase partitioning system canisolate a species from a mixture, or cause a species to partition atleast in some way differently between the phases. Where a two-phasesystem is described herein, it is to be understood that more phases canbe used.

“Aqueous multi-phase system,” as used herein, refers to an aqueoussystem which includes greater than one aqueous phase in which an analytespecies can reside, and which can be used to characterize the structuralstate of the analyte species according to the methods described herein.For example, an aqueous multi-phase system can separate at equilibriuminto two, three, or more immiscible phases. Aqueous multi-phase systemsare known in the art, and this phrase, as used herein, is not meant tobe inconsistent with accepted meaning in the art. Examples of variousaqueous multi-phase systems, and their compositions, are described morefully below.

“Cumulative spectral information” means spectral information includinginput from a plurality of species, e.g., a spectrum (which can be a massspectrum report) representing a mixture of species.

An “interacting component” means a component, such as a phase of amulti-phase system, or a component of a chromatography column or othercomposition able to cause separation, that can interact with a speciesand provide information about that species (for example, an affinity forthe species). Multiple interacting components, exposed to a species, candefine a system that can provide a “relative measure of interaction”between each component and the species. An interacting component can besolid or liquid, aqueous or non-aqueous, can be polymeric, organic (e.g.a protein, small molecule, etc.), inorganic (e.g. a salt), a surfactant,or the like, or any combination thereof. A set of interacting componentscan form a system useful in and in part defining any experimental methodwhich is used to characterize the structural state of a species such asan analyte species according to the methods described herein. Typically,a system of interacting components can measure the relative interactionbetween the species and at least two interacting components.

An aqueous multi-phase system is an example of a system of interactingcomponents, and it is to be understood that where “aqueous system” or“aqueous multi-phase system” is used herein, this is by way of exampleonly, and any suitable system of interacting components can be used.Where aqueous two-phase and aqueous multi-phase systems are describedherein, it is to be understood that other systems, as used herein,systems analogous to those comprising only aqueous solutions orsuspensions can be used. For example, an aqueous two-phase system caninclude non-aqueous components in one or more phases that are not liquidin character. In this aspect, multi-phase systems also refers to relatedtechniques that rely on differential affinity of the biomolecule to onemedia versus another, where the transport of the biomolecule between onemedium and, optionally, another medium occurs in an aqueous ornon-aqueous environment. Examples of such multi-phase systems include,but are not limited to, HPLC columns or systems for liquid-liquidpartition chromatography or other forms of chromatography, as are knownto those of ordinary skill in the art, where one phase may be a solidphase and another phase may be a liquid which carried species proximtethe solid phase, and different affinity among various species for thesolid and/or liquid phases can cause separation. It should be understoodthat the invention is not limited to aqueous multi-phase systems; insome cases, a system having a single, non-partitioned phase may be used;for example, two or more interacting components may define a solventcontaining a plurality of species, and the components may be at leastpartially separated (although there may not necessarily be a cleardivision between the separated components, e.g., with respect toconcentration, etc.)

“Relative measure of interaction,” with reference to a particularspecies as used herein, means the degree to which the species interactswith another species or with a phase of a multi-phase system in arelative sense. For example, a particular species may have a greateraffinity for one phase of a multi-phase system rather than another phaseor phases, and the degree to which it interacts with or resides in, thatphase, as opposed to other phases, defines its relative measure ofinteraction. Relative measures of interaction, in the context of thepresent invention, are generally determined in a ratiometric manner,rather than an absolute manner, although in some cases, the absolutemanner can be used. As a non-limiting example, where a species caninteract with each phase of a two-phase system but resides morepreferably in one than the other, the present invention typically makesuse of information as to the ratio of concentration of the species ineach of the two phases, or in one of the phases and the original sample,but not necessarily of the absolute concentration of the species ineither phase. In other cases, the interaction can be an interactionbased not upon residence of a particular species within a particularsolvent or fluid carrier, but upon interaction with a solid surface,such as a solid phase of a chromatography column, where the relativemeasure manifests itself in elution time, and/or can involve geometricor spatial interaction such as a particular species interaction with aporous substrate as opposed to that of a different species or adifferent substrate. In some cases, the relative measure of interactionincludes more than one type of interaction.

“Partition coefficient,” as used herein, refers to the coefficient whichis defined by the ratio of chemical activity or the concentrations of aspecies in two, three, or more phases of a multi-phase system atequilibrium, or the ratio of chemical activity or the concentration of aspecies in one phase of a multi-phase system at equilibrium and thecorresponding species in the original sample. For example, the partitioncoefficient (“K”) of an analyte in a two-phase system is defined as theratio of the concentration of analyte in the first phase to that in thesecond phase. For multi-phase systems, there may be multiple partitioncoefficients, where each partition coefficient defines the ratio ofspecies in first selected phase and a second selected phase. It will berecognized that the total number of partition coefficients in anymulti-phase system is typically equal to the total number of phasesminus one. As used herein, the term “partition coefficient” also canrefer to the ratio of the peak height at a specific m/z (mass-to-chargeratio) location from the analysis of the mixture of species in the firstphase of a two-phase system to the corresponding height at the samespecific m/z location from the analysis of the mixture of species in thesecond phase of the same two-phase system, or to the peak height ratioat a specific elution time in a chromatographic analysis between samplesfrom two, three, or more different phases of such a system.

For heterogeneous phase systems, an “apparent partition coefficient,” asused herein, refers to a coefficient which describes informationobtained from alternative techniques that is correlated to the relativepartitioning between phases. As a non-limiting example, if theheterogeneous two-phase system used is an HPLC column or otherchromatography column, this “apparent partition coefficient” can be therelative retention time for the analyte. It will be recognized by thoseof ordinary skill in the art that the retention time of an analyte in achromatography column, in many cases, reflects the average partitioningof the analyte between a first, mobile phase and a second, immobilephase. Also, it will be recognized that other, similarly determinableproperties of analytes can also be used to quantify differences inphysical properties of the analytes (e.g. in other techniques) and are,therefore, suitable for use as apparent partition coefficients.

“Bind,” as used herein, means the well understood receptor/ligandbinding, as well as other nonrandom association between an a biomoleculeand its binding partner. “Specifically bind,” as used herein, describesa binding partner or other ligand that does not cross-reactsubstantially with any biomolecule other than the biomolecule orbiomolecules specified. Generally, molecules which preferentially bindto each other are referred to as a “specific binding pair.” Such pairsinclude, but are not limited to, an antibody and its antigen, a lectinand a carbohydrate which it binds, an enzyme and its substrate, and ahormone and its cellular receptor. Examples of binding mechanismsinclude, but are not limited to, covalent, ionic, van der Waals,hydrogen, or the like.

As generally used, the terms “receptor” and “ligand” are used toidentify a pair of binding molecules. Usually, the term “receptor” isassigned to a member of a specific binding pair, which is of a class ofmolecules known for its binding activity, e.g., antibodies. The term“receptor” is also preferentially conferred on the member of a pair thatis larger in size, e.g., on lectin in the case of thelectin-carbohydrate pair. However, it will be recognized by those ofskill in the art that the identification of receptor and ligand issomewhat arbitrary, and the term “ligand” may be used to refer to amolecule which others would call a “receptor.” The term “anti-ligand” issometimes used in place of “receptor.”

“Molecule-molecule interaction,” such as a biomolecule-biomoleculeinteraction, a protein-protein interaction, and the like, means aninteraction that typically is weaker than “binding,” i.e., aninteraction based upon hydrogen bonding, van der Waals binding, Londonforces, or other non-covalent interactions that contribute to anaffinity of one molecule for another molecule, which affinity can beassisted by structural features such as the ability of one molecule toconform to another molecule or a section of another molecule.Molecule-molecule interactions can involve binding, but need not.

“Biomolecule,” as used herein, means a molecule typically derived froman organism, and which typically includes building blocks includingnucleotides, and the like. Non-limiting examples include peptides,polypeptides, proteins, protein complexes, nucleotides,oligonucleotides, polynucleotides, nucleic acid complexes, saccharides,oligosaccharides, carbohydrates, lipids as well as combinations,enantiomers, homologs, analogs, derivatives and/or mimetics thereof.

“Species,” as used herein, refers to a molecule or collection ofmolecules. For example, an inorganic chemical, an organic chemical, abiomolecule, or the like may be a species. In the present invention,species generally are biomolecules.

“Corresponding species,” as used herein, means at least two differentspecies that are identical chemically or, if they differ chemicallyand/or by molecular weight, differ only slightly. Non-limiting examplesof corresponding species include structural isoforms of proteins,proteins or other molecules that are essentially identical but thatdiffer in binding affinity with respect to another species or pluralspecies, have different higher-order structure, e.g., differing insecondary or tertiary structure but not differing or not differingsignificantly in chemical sequence. In general, corresponding speciesare species that may be arranged differently (isoforms, isomers, etc.)but are composed of the same or essentially the same chemical buildingblocks.

“Detectable,” as used herein, refers the ability of a species and/or aproperty of the species to be discerned. One example method of renderinga species detectable is to provide further species that bind or interactwith the first species, where the species comprise(s) a detectablelabel. Examples of detectable labels include, but are not limited to,nucleic acid labels, chemically reactive labels, fluorescence labels,enzymatic labels and radioactive labels.

As used herein, the term “determining” generally refers to the analysisof a species, for example, quantitatively or qualitatively, and/or thedetection of the presence or absence of the species. “Determining” mayalso refer to the analysis of an interaction between two or morespecies, for example, quantitatively or qualitatively, and/or bydetecting the presence or absence of the interaction.

“Mimetic,” as used herein, includes a chemical compound, an organicmolecule, or any other mimetic, the structure of which is based on, orderived from, a binding region of an antibody or antigen. For example,one can model predicted chemical structures to mimic the structure of abinding region, such as a binding loop of a peptide. Such modeling canbe performed using standard methods (see, for example, Zhao et al., Nat.Struct. Biol. 2: 1131-1137 (1995)). The mimetics identified by methodssuch as this can be further characterized as having the same bindingfunction as the originally identified molecule of interest, according tothe binding assays described herein. Alternatively, mimetics can also beselected from combinatorial chemical libraries in much the same way thatpeptides are. See, for example, Ostresh et al., Proc. Natl. Acad. Sci.U.S.A. 91: 11138-11142 (1994); Dorner et al., Bioorg. Med. Chem. 4:709-715 (1996); Eichler et al., Med. Res. Rev. 15: 481-96 (1995);Blondelle et al., Biochem. J. 313: 141-147 (1996); or Perez-Paya et al.,J. Biol. Chem. 271: 4120-6 (1996).

“Solid support,” as used herein, means the well-understood solidmaterial to which various components of the invention are physicallyattached, thereby immobilizing the components of the present invention.The term “solid support,” as used herein, means a non-liquid substance.A solid support can be, but is not limited to, a membrane, sheet, gel,glass, plastic or metal. Immobilized components of the invention may beassociated with a solid support by covalent bonds and/or vianon-covalent attractive forces such as hydrogen bond interactions,hydrophobic attractive forces and ionic forces, for example.

“Structure,” “structural state,” “configuration,” or “conformation,” asused herein, all refer to the commonly understood meanings of therespective terms, for example, as they apply to biomolecules such asproteins and nucleic acids, as well as pharmacologically active smallmolecules. In different contexts, the meaning of these terms will vary,as is appreciated by those of skill in the art. The structure orstructural state of a molecule refers generally not to the buildingblocks that define the molecule but the spatial arrangement of thesebuilding blocks. The configuration or confirmation typically definesthis arrangement. For instance, the use of the terms primary, secondary,tertiary, or quaternary, in reference to protein structure, haveaccepted meanings within the art, which differ in some respects fromtheir meaning when used in reference to nucleic acid structure (see,e.g., Cantor and Schimmel, Biophysical Chemistry, Parts I-III). Unlessotherwise specified, the meanings of these terms will be those generallyaccepted by those of skill in the art.

“Physiological conditions,” as used herein, means the physical,chemical, or biophysical state of an organism. As most typically used inthe context of the present invention, physiological condition refers toa normal (e.g., healthy in the context of a human or other organism) orabnormal (e.g., in a diseased state in the context of a human or otherorganism) condition.

“Pattern,” as used herein, means a sequence of physical properties ofspecies, or a combination of physical properties and other properties.

“Corresponding pattern,” as used herein, means a second pattern that istypically obtained from a different sample of biological system orsystems, and is comprised of the same sequence of physical or otherproperties, such that each location in the sequence possesses the samevalue of the descriptor, whether numerical or categorical in nature.

“Marker” as used herein, is a pattern of physical properties of species(e.g., a spectrum of m/z values obtained from mass spectrometryexperiments of a mixture of species) that can be a carrier ofinformation regarding the structural or physiological state of abiological environment within which it resides. A pattern of suchphysical properties can also be mathematically or statisticallyprocessed, condensed, transformed, or represented otherwise. A“processed marker,” as used herein, is such a processed pattern, but asused herein, marker is used to alternatively designate the pattern ofphysical properties or the processed pattern of such properties,depending on the particular context. A marker can exhibit at least twodifferent properties or values of a specific property or properties(e.g., structural conformation, binding affinity for another species,etc., but not solely different amounts of the species) that correspondto and that represent information regarding the two or morephysiological states of environments within which they reside. Forexample, a marker may be a pattern obtained from a series of proteins,some of which are structurally modified between a first staterepresentative of a healthy system within which it resides, and a secondstructural state (e.g., different conformations) representative of adisease system within which it resides. As used herein, a marker is alsoa comparative pattern that can be a carrier of information regarding aphysiological state of a biological environment within which it resides,and/or a combination of such patterns and other information related toother properties.

“Spectral data” means any information, whether visible, recorded onpaper, recorded electronically, or the like, relating to application ofone or more spectral techniques to a sample, such as mass spectralinvestigation, infrared spectral investigation, UV and/or visiblespectroscopy, NMR spectroscopy, or any other sequence of data obtainedfrom sample analysis, in which the independent variable could includewavelength, elution time, etc.). Such spectral analysis could beperformed by those of ordinary skill in the art using no more thanroutine skill.

Spectral data is one example of a data pattern, which is not limited totechniques in which the sample is interrogated vs. electromagneticwavelength. A data pattern includes any sequence of analysis dataobtained by other techniques, e.g., by chromatographic techniques thatproduce data streams vs. elution time rather than vs. wavelength, byNMR, etc.

“Comparing,” in the context of spectra or spectral data (or other datapattern), means any type of comparison of any section or sections of thespectral data (or other data pattern). For example, two typicalprintouts from a mass spectrometer can be compared side-by-side and ahuman can observe the height of one or more mass spectral peaks of eachspectrum and make a notation as to comparison between those peaks, orcan observe numbers associated with computer analysis representingmass-to-charge ratios associated with such peaks and compare thesenumbers. Alternatively, a computer can be programmed to compare variousmass-to-charge peaks of various mass spectra to each other and toproduce a comparative spectrum, or comparative spectral information,representing such comparison. Comparative spectral information maydefine, therefore, a number representing a difference in two spectra orother data patterns, and/or the number can define a ratio of the peakheight or extent of two different spectra at a particular mass-to-chargeratio location (with the example of mass spectral data). Alternatively,a comparative spectrum can record differences in two or more datapatterns at a plurality of spectral data points, that is, in the case ofmass spectra a plurality of peaks at specific mass-to-charge ratios,recorded either as differences or ratios between the two spectra. As afurther example, all data points of two mass spectra can be compared,either as differences between peaks at specific mass-to-charge ratios ofeach spectra or ratios between each, and displayed as a series ofnumbers or as a new spectral printout, or displayed any other way, wherean observer or a machine (e.g., processor) can observe and/or analyzethis data and thereby observe and/or record differences betweendifferent samples from which each spectrum was derived. All suchspectral data and comparative spectra can be obtained with or withoutanalysis of the data of the type that would lead to identification ofany one or more species of the sample from which the spectral data wasobtained. The data pattern could also be first mathematically processed,transformed to another domain, e.g., using Fourier or wavelettransforms, and the processed or transformed data could be compared toother spectra using techniques known to those skilled in the art,including simple mathematical operations, data reduction techniques suchas eigenvalue analysis and alike. The use of comparison of spectra andthe term “comparative spectrum” generally involves at least two datapatterns of the same sample, at least one of which was obtained byinteracting the sample with a multi-phase system. The action ofcomparison generally does not involve simply normalizing the datapattern, e.g., to obtain a baseline as customarily done in spectrometricor chromatographic analyses. The comparative spectrum is also generallyderived in such a way that it does not depend on the concentration ofspecies comprising the original sample, and may not necessarily carryinformation regarding the concentration or levels of abundance of thespecies in the original sample.

“Comparative spectral data” can be similarly derived from IR Spectra,UV/Vis spectra, and the like, as well as from a sequence of data pointsin time obtained, e.g., from a chromatographic elution profile. Those ofordinary skill in the art are familiar with comparative spectral data inconnection, at least, with UV/Vis and IR Spectra, and a variety oftechniques for recordation and/or display of such data. It is to beemphasized that, in connection with the invention, spectral data and/orcomparative spectral data can be obtained and/or derived, in connectionwith a particular sample or samples, at any one or a number of datapoints associated with the spectra (any number of mass-to-charge ratios,wavelengths, elution times, etc.).

Embodiments

Recent advances in the study of spectral analysis, e.g.,mass-spectrometry patterns of proteins and mixtures obtained from serumor other biological fluid, have demonstrated that one does not need toexplicitly identify the proteins that differ between two samples to beable to distinguish between them. (It is to be understood that, anywherein this application that a particular spectral technique is described,e.g., mass spectrometry, the particular spectral technique can besubstituted by any other spectral or chromatographic technique, forexample, as disclosed herein. In one particular set of embodiments ofthe invention, mass spectrometry is employed.) Thus, in some aspects,using signal pattern techniques, the differences in spectra can beexpressed in mathematical terms that capture the pattern of the mixture(a “data pattern”) without requiring their identification. Instead ofidentifying differences between samples by detecting changes in theconcentration levels of specific proteins (biomarkers) or other species,the patterns representing the entire mixtures of proteins or otherspecies in the samples are compared and subsequently used to classifythe samples. These techniques are especially sensitive to changes inconcentration levels that are not related to the, e.g., pathological orphysiological changes that correspond to the samples under analysis. Forcertain applications, e.g., diagnostics of relatively rare diseases ingeneral populations, the required level of specificity and sensitivitycan be very high. Such high performance levels, coupled with inherentsensitivity of pattern recognition techniques to unrelated changes inconcentration levels in the samples, can be addressed using certainsystems and methods of the present invention. Pattern recognition-basedmethods as described in the present invention are inherently insensitiveto absolute levels of expression, and may result in much more reliablepatterns. Moreover, such patterns could be more closely correlated withthe underlying differences between the samples. As mentioned, suchcomparative techniques are not limited only to mass spectroscopy data,but can also be applied to any data pattern that is produced inassociation with one or more species of a sample, for example, NMRspectroscopy, UV and/or visible spectroscopy, chromatography (e.g.,liquid chromatography or HPLC), GPC, ELIZA, etc.

The state of a molecule or other species (e.g., a molecular aggregate, amulti-subunit protein, etc.), such as a biomolecule, can be affected bymany factors including, but not limited to, changes in the chemicalstructure (e.g., addition, deletion or substitution of amino acids inproteins, covalent modification by chemical agents or cleavage bychemical or thermal degradation, addition or deletion of carbohydratesto the structure, etc.), interactions with one or more other speciessuch as biomolecules or ligands, or the like. The evaluation ofdifferent states can be used as one method of determining the potentialeffectiveness of different molecules (or other species), condition ofthe molecules, condition or state of an environment (e.g., a mixture ofspecies) within which the molecule or species resides, and the like.

The present invention involves, in some embodiments, the investigationof the state of molecules. The invention is described in the context ofstudies involving biomolecules and/or molecules able to interact withbiomolecules, but the invention can apply to essentially any molecularspecies and/or interaction, whether biological, biochemical, chemical,or other species, and those of ordinary skill in the art will understandhow the invention can be used in the context of non-biologicalmolecules. It is to be understood that whenever “biomolecules” is usedin the description of the invention, any non-biological molecule canalso be used or studied.

In one aspect, the present invention involves techniques for determininginformation about the composition of a mixture of biomolecules (or otherspecies) and/or molecules which interact with biomolecules. The mixturemay originate from biological material, such as human clinical sample orother biological fluid, tissue, cells, a subject, etc., and/or themixture may be a synthetic mixture. The mixture can come from abiological system which, as used herein, means a human or non-humanmammal, including, but not limited to, a dog, cat, horse, cow, pig,sheep, goat, chicken, primate, rat, and mouse, other animals (e.g.,frog), or a bacteria, virus, fungus, or of plant origin. The mixture maybe taken from any suitable source within the human or other animal, forexample, from tissue biopsies, whole blood, serum or other bloodfractions, urine, ocular fluid, saliva, cerebro-spinal fluid, fluid orother samples from tonsils, lymph nodes, needle biopsies, etc.

The invention also relates, in some cases, to developing and determiningcharacteristics (quantitative and/or qualitative) of a mixture that areobtained, for example, via processing using multi-phase partitioning orother separation techniques as described herein (e.g., chromatography),which can reflect certain structural and/or functional characteristicsof biomolecules or molecules that interact with biomolecules in theoriginal mixture. These characteristics can be used, for example, forestablishing relationships between the composition of the mixture andthe physiological state of the biological source of the mixture e.g.,the state of health or disease of a subject. These characteristics canalso be used to design experimental conditions for subsequentfractionation of the mixtures into subsets enriched in the molecule(s)of interest for the purpose of the analysis, while simultaneouslyreducing the total number of different molecule(s) in some cases. Theseparation may be full or partial, i.e., one or more species is presentin a higher concentration in the subset, relative to the overall sample,but other species may still be present in the subset. The systems andmethods of the present invention can also be useful for detecting,classifying, and/or predicting changes in a mixture of biomolecules ormolecules that interact with biomolecules. For example, the mixture maybe a synthetic mixture, or a mixture associated with a particulardisease or physiological state of a living organism, cells, tissues, orbiological liquids. The systems and methods of the present invention canalso be used to detect changes to a one or more molecules orbiomolecules in a biological mixture, and these changes could further beused, in some embodiments, to detect and classify a diagnostic that isrelated to such changes. However, in one embodiment, the systems andmethods of the present invention are not used to simply remove a subsetof species of the original mixture prior to analysis, even though suchfractionation could be accomplished as a pre- (or post-) processing stepof the mixture prior to (or subsequent to) interacting the mixture witha multi-phase system and deriving a comparative spectrum in the mannerdescribed herein.

Examples of such changes in a mixture can be the differences inproperties of a pattern of species of the mixture, such as those relatedto differences in the species conformation, structure, and/orinteraction tendency with respect to another molecule or molecules(e.g., its binding affinity or other interaction characteristic withrespect to another molecule or molecules, or other species). Forexample, if the mixture includes proteins or other biomolecules, suchchanges may be induced through primary sequence modification, bydegradation of the proteins or other biomolecules through chemical,thermal, or other degradation mechanisms, by interaction with othermolecules and/or biomolecules, by interaction with low molecular weightcompounds (e.g., hormones, peptides, vitamins, cofactors, etc.), bychanges in the relative content or concentration of the constituents ofthe mixture, by reactions such as enzymatic reactions, by specificchanges such as phosphorylation or glycosylation, etc. The systems andmethods of the present invention can be used, in some cases, to detect,analyze and/or characterize biological species, including but notlimited to, polypeptides, proteins, carbohydrates, nucleic acids,polynucleotides, lipids, and/or sterols, and mixtures or derivativesthereof, e.g., for the purpose of detection of, or onset of, aparticular disease or physiological state, monitoring its progress,treatment, etc.

Comparison and classification steps involved in the invention can makeuse of additional information not necessarily related to (not directlyderived from) the analytical methods of the invention. For example,blood pressure, temperature, blood glucose level, and/or essentially anyother measurable physiological condition can be used in conjunction withtechniques of the invention to analyze one or more physiologicalconditions.

In some embodiments of the invention, a plurality of species (molecules,biomolecules, etc.) is exposed to at least first and second interactingcomponents, which may at least partially separate or “partition” theplurality of species, e.g., into a first portion and a second portionhaving a different composition than the first portion. For example, thefirst portion may be enriched in a first species (or a firstconformation of a first species), while the second portion may bedeficient in the first species or conformation. Such separation may bepartial or total, in some cases. In some cases, the system is apartitioning system, as disclosed herein. Non-limiting examples includeaqueous/non-aqueous partitioning systems, aqueous multi-phasepartitioning system, liquid chromatography, HPLC, column liquid-liquidpartition chromatography (LLPC), a heterogeneous two-phase system, amultiphase heterogeneous system, etc.

Multiple partitioning and/or other separation steps can take place insome embodiments of the invention so that additional information and/orsensitivity can be obtained. For example, prior to determiningcomparative patterns of species in each of two or more differentmixtures, and following partitioning of both mixtures in two, three, ormore partitioning systems of identical (or nearly identical)composition, a quantity of the first and/or the second interactingcomponents of both systems containing the mixtures can be furtherintroduced into a second set of two identical (or nearly identical)systems with at least two interacting components. Then, partitioning ofboth second sets of systems for both mixtures can take place, andcomparative patterns of species in each mixture can be determined andused as described herein. As another non-limiting example, a mixture mayfirst be subjected to chromatography, followed by partitioning (or viceversa).

It will be recognized by those of ordinary skill in the art that thesebiological species can be found in any suitable form, for example, inthe form of extracts from natural sources, biological liquids, cell andtissue samples, bacteria, virus or fungus, collections of moleculesgenerated by combinatorial chemical or biochemical techniques andcombinations thereof, synthetically created, etc.

In one embodiment, the present invention provides a method to determinecertain conditions under which variations among samples representingdifferent compositions (or mixtures of species) could be detected, i.e.,determining a set of criteria and/or system components as a “tool,” or apart of a tool, to determine information. For example, the ability of amulti-phase system to determine a comparative pattern of species canserve as an important tool or component of such a tool. Specifically, asone non-limiting example, the partitioning of the constituents of asample between two phases having different chemical or biochemicalaffinities or other characteristics, such as solvent structures, mayseparate the constituents by their relative affinity for media ofdifferent properties or composition. This separation technique thus caninclude or, alternatively, can be unlike those typically used inproteomics or similar techniques, e.g., 2D gel electrophoresis, in whichcharge and size differences are the two dimensions used to separate theconstituents of a sample. In some cases, e.g., for many applications inproteomics, the present invention provides the ability for performingsequential and/or serial partitioning, with either the same of differentconditions, which may result in additional amplification or differencesin the fractionated samples. These patterns of physical properties ofspecies comprising such fractions may be further analyzed in some casesusing techniques such as mass spectrometry. However, in the context ofthe present invention, it is not necessarily the intent of suchoperations to simply fractionate or remove some species comprising themixture, but to provide for means to derive a comparative spectrum thatcan be independent of information regarding the concentration orabundance levels of the original species in the mixture.

As mentioned herein, aqueous multi-phase (e.g., two-phase) partitioningsystems are well-suited for use in many embodiments of the invention,but other partitioning systems can be used as well, according to otherembodiments. Thus, where “aqueous two-phase partitioning” or “aqueousmulti-phase partitioning” is used, it is to be understood that othersystems can be used, for example, aqueous/non-aqueous two-phasepartitioning, non-aqueous/non-aqueous two-phase partitioning, liquidchromatography, etc. Partitioning of a biopolymer in aqueous two-phasesystems may depend on factors such as its charge, size,three-dimensional structure, type, topography of chemical groups exposedto the solvent, etc. For instance, changes in the 3D structure of areceptor induced by some effect, e.g., by binding of a ligand bindingand/or by structural degradation, also can change the topography ofsolvent accessible chemical groups in the biomolecule, or both thetopography and the type of the groups accessible to solvent. One resultof these changes may be an alteration in the partition behavior of thebiomolecule and/or the ligand-bound receptor, according to certainembodiments of the invention.

In some cases, the level of concentration of biomolecules in biologicalsamples is dependent upon genotyping or reasons other than those relatedto the physiological condition under investigation. Thus, identificationof differences in biomolecules attributable to diseased verses normalstates may necessitate using a statistically significant number ofsamples to negate the effect of natural genetic or other variations insome embodiments of the invention. In some cases, the effect of geneticor other variability, leading to under- or overexpression, can beseparated (e.g., fractionated) from differences to biomolecules that aretraced to their diseased versus normal states. This separation can beachieved by subjecting a sample or other mixture of species containingbiomolecules or other molecules to partitioning or separation in one ormore different systems, and determining a comparative pattern of speciesin the sample/mixture with various components of the system(s),according to various embodiments. This can be accomplished, e.g., byseparating and/or fractionating, using conventional techniques, the twointeracting components of each sample, calculating the pattern ofpartition coefficients calculated for each species in the diseased andnormal samples, and utilizing such pattern for further analysis. Asspecific non-limiting examples, obtaining a comparative pattern caninvolve fractionating or separating at least a portion of the firstportion and second portion (and/or more portions) of the system. Thisfractionating or separation can involve techniques includingelectrophoresis such as one-dimensional electrophoresis, two-dimensionalelectrophoresis, liquid or other chromatography, direct or subsequentanalysis performing mass spectrometry on at least a portion of thefirst, second (and, alternately, more portions) of the system, or thelike, and in some cases, can involve a point-by-point basis ofcomparison. Other techniques include other spectrographic techniques(e.g., UV, visible, IR, Raman, etc.), etc. Different partitioncoefficients may not be related to the absolute level of expression ofeach species, but instead, may be related to changes to the structure,binding to other molecules or other changes of relevance to theirbiological effects, etc. Thus, the present invention provides, in oneset of embodiments, methods for the identification of changes tobiomolecules in a biological mixture that may be inherent in theirstructure and thus more closely to their function and not their absolutelevel, and in some cases without necessarily requiring a largestatistical number of samples to negate the effect of individualvariability in the expression levels.

The use of a pattern of partition coefficient values that is obtainedfrom multiple systems (a “signature”) can be used to enhance thespecificity of certain methods of the invention. In yet otherembodiments, partitioning of the samples in multiple systems andperforming the steps above, then observing the pattern of values for oneor more biomolecules, can provide another way to constructing asensitive and specific diagnostics method.

In some embodiments, such changes may be detected using other systemsand methods which have an underlying dependence upon the topographyand/or the types of solvent accessible groups. Examples of such othermethods include, but are not limited to, column liquid-liquid partitionchromatography (LLPC), a heterogeneous two-phase system, a multiphaseheterogeneous system, etc. In some cases, an apparent partitioncoefficient may be generated that expresses the relative changes in theaverage partitioning between a first and a second phase. For example, inLLPC, the retention volume of a receptor may be used as the apparentpartition coefficient.

Aqueous two-phase systems are well-known to those of ordinary skill inthe art and can arise in aqueous mixtures of different water-solublepolymers or a single polymer and a specific salt. When two or morecertain polymers, e.g., dextran (“Dex”) and polyethylene glycol (“PEG”),or one or more certain polymers and one or more inorganic salts, e.g.polyvinylpyrrolidone (“PVP”) and sodium sulfate, are mixed in waterabove certain concentrations, the mixture can separate into twoimmiscible aqueous phases under certain conditions. There may be adiscrete interfacial boundary separating two phases, for example, suchthat one is rich in one polymer and the other phase is rich in the otherpolymer or the inorganic salt. The aqueous solvent in one or both phasesmay provide a medium suitable for biological or other species. Two-phasesystems can also be generalized to multiple phase system by usingdifferent chemical components, and aqueous systems with a dozen or morephases are known in the art and can be used in connection with theinvention.

When a species is introduced into such a two-phase system, it maydistribute between the two phases. In this and other systems (e.g.,multiphase systems having three or more such phases), the species can befound at different concentrations within each phase, or can be at thesame concentration within each phase. Partitioning of a solute can becharacterized by the partition coefficient “K,” defined as the ratiobetween the concentrations of the solute the two immiscible phases atequilibrium. It has previously been shown that phase separation inaqueous polymer systems may result from different effects of twopolymers (or a single polymer and a salt) on the water structure (see,e.g., B. Zavlavsky, Aqueous Two-Phase Partitioning: Physical Chemistryand Bioanalytical Applications, Marcel Dekker, New York, 1995). As theresult of the different effects on water structure, the solvent featuresof aqueous media in the coexisting phases can differ from one another.The difference between phases may be demonstrated by techniques such asdielectric, solvatochromic, potentiometric, partition measurements, orthe like.

The basic rules of solute partitioning in aqueous two-phase systems havebeen shown to be similar to those in water-organic solvent systems(which can also be used as systems in the present invention). However,what differences do exist in the properties of the two phases in aqueouspolymer systems are often very small, relative to those observed inwater-organic solvent systems, as would be expected for a pair ofsolvents of the same (aqueous) nature. The small differences between thesolvent features of the phases in aqueous two-phase or multi-phasesystems can be modified so as to amplify the observed partitioning thatresults when certain structural features are present.

The polymer and/or salt compositions of each of the phases may dependupon the total polymer and/or salt composition of an aqueous two-phasesystem. The polymer and/or salt composition of a given phase, in turn,can govern the solvent features of the aqueous media in this phase.These features include, but are not limited to, dielectric properties,solvent polarity, ability of the solvent to participate in hydrophobichydration interactions with a solute, ability of the solvent toparticipate in electrostatic interactions with a solute, and hydrogenbond acidity and basicity of the solvent. All these and other solventfeatures of aqueous media in the coexisting phases may be manipulated byselection of polymer and salt composition of an aqueous two-phasesystem. These solvent features of the media may govern the sensitivityof a given aqueous two-phase system toward a particular type of solventaccessible chemical groups in the receptor. This sensitivity, type, andtopography of the solvent accessible groups in two different proteins,for example, can determine the possibility of separating proteins in agiven aqueous two-phase system.

In some cases, a particularly sensitive system may be required, e.g., asystem that is very sensitive to two very similar species, or a systemable to detect differences in conformation of a single species, etc.This sensitivity may be of importance when, for example, subtledifferences are being detected between the conformational changes in areceptor induced by binding of closely related chemical compounds. Thepresent invention provides, in some embodiments, efficient andsuccessful systems and methods for screening compositions to identifyand/or amplify differences between the compositions of two mixtures. Byutilizing a wide variety of different conditions to screen eachmolecule, as described herein, different partitioning or separationbehavior may be obtained reliably, without the need to fully understandthe underlying theory of aqueous two-phase partitioning, or any of theother related or substitutable separation techniques.

Biomolecules such as proteins, nucleic acids, etc. may be distributedbetween the two or more phases when placed into such a system. Forexample, in the case where phase-forming polymers are used, solutionscomprising one or more of the two polymers and the biomolecule may bemixed together such that both phase-forming polymers and the biomoleculeare mixed. The resulting solution is resolved and a two-phase system isformed. Optionally, centrifugation can be used to enhance separation ofthe phases. In yet another embodiment, the partitioning may be conducedin a microfluidic device, in which two liquid streams are brought intoclose contact in a narrow channel thus facilitating partitioning ofspecies without requiring agitation and centrifugation. It will berecognized by those of ordinary skill in the art that partitioningbehavior of a biomolecule may be influenced by many variables, such asthe pH, the polymers used, the salts used, factors relating to thecomposition of the system, as well as other factors such as temperature,volume, etc. Optimization of these factors for desired effects can beaccomplished by routine practice by those of ordinary skill in therelevant arts, in combination with the current disclosure.

Evaluation of data from partitioning of a biomolecule or other speciescan involve use of the partition coefficient(s), in some embodiments.For example, the partition coefficient of a protein can be taken as theratio of the protein in first phase to that in the second phase in abiphasic system. When multiple phase systems are formed, there can bemultiple independent partition coefficients, each of which can bedefined between any two phases. It will be recognized that the partitioncoefficient for a given biomolecule or other species of a givenconformation will be a constant if the conditions and the composition ofthe two-phase system to which it is subjected remain constant. Thus, forexample, if changes are observed in the partition coefficient for aprotein upon addition of a potential binding partner, these changes canbe presumed to result from changes in the protein structure caused byformation of a protein-binding partner complex. The partitioncoefficient in such cases is a specifically mathematically definedquantity, and the term includes coefficients representing the relativemeasure of interaction between a species and at least two interactingcomponents. It should also be recognized that differences betweenpartition coefficients of corresponding species in two or more mixturescould indicate, in addition to potential structural changes, binding orlack of binding of such species to other species in the mixtures. Thepresent invention makes specific use of patterns of partitioncoefficients and not necessarily their individual counterparts forpurposes described herein.

In a non-limiting example of one partitioning system, aqueous multiphasesystems are known to be formable from a variety of substances. Forexample, in order to determine the partition coefficient of a protein(or a mixture of a protein with another compound) to be analyzed,concentrated stock solutions of all the components (polymer 1, e.g.,dextran; polymer 2, e.g., PEG, polyvinylpyrrolidone, salts, etc.) inwater can be prepared separately. The stock solutions of phase polymers,salts, and the protein mixture can be mixed in the amounts andconditions (e.g., pH from about 3.0 to about 9.0, temperature from about4° C. to 60° C., salt concentration from 0.001 mol/kg to 5 mol/kg)appropriate to bring the system to the desired composition andvigorously shaken. The system can then be allowed to equilibrate(resolution of the phases). Equilibration can be accomplished byallowing the solution to remain undisturbed, or it can be accelerated bycentrifugation, e.g., for 2-30 minutes at about 1000 g to about 4000 g,or higher in some cases. Aliquots of each settled (resolved) phase canbe withdrawn from the upper and/or lower phases (or from one or morephases, if multiple phases are present). The concentration ofmolecule(s) or other species can then be determined for each phase.

Different assay methods may be used to determine the relative measuresof interaction between species and interacting components in variousembodiments, e.g. in the form of the concentration of the biomoleculesin each phase of a multi-phase system. The assays will often depend uponthe identity and type of biomolecule or other species present. Examplesof suitable assay techniques include, but are not limited to,spectroscopic, immunochemical, chemical, fluorescent, radiological, andenzymatic assays. When the biomolecule is a peptide or protein, thecommon peptide or protein detection techniques can be used. Theseinclude, but are not limited to, direct spectrophotometry (e.g.,monitoring the absorbance at 280 nanometers) and dye binding reactionswith Coomassie Blue G-250 or fluorescamine, o-phthaldialdehyde, or otherdyes and/or reagents. Alternatively, if the protein is either anantibody or an antigen, certain immunochemical assays can be used insome cases. In the case of mass spectrometry, the peak height at aspecific m/z spectral location may be proportional to the concentrationof the specific protein in some instances, or the peak height at aspecific elution time may be proportional to the same.

The concentration of the biomolecule(s) or other species in each phase,or in one phase and the original sample, can be used to determine thepartition coefficient of the sample under the particular systemconditions, in some embodiments of the invention. Since the partitioncoefficient reflects only the ratio of the two concentrations, theabsolute values may not be required. It will be recognized that this canallow certain analytical procedures to be simplified, e.g., calibrationcan be eliminated in some instances. It also may have significantadvantages for negating the effect of natural variability in theabsolute concentration of proteins in samples obtained from, e.g.,biological systems, when comparing two or more samples, thus focusing onthose changes detected as differences in the partition coefficientrelevant to changes to the structure of the individual species in thesamples.

It should be recognized by those skilled in the art that the steps inabove description of obtaining the partition coefficient could besubstituted by others. Depending on the size, volumes, amount of thebiomolecule, detection system, discrete or continuous operation usingeither liquid-liquid or liquid-solid partioning, chromatography, orother processes that effectively result in results described herein maybe used. Such modifications and different processes do not limit thescope of the present invention.

The partition coefficient can also be compared with other partitioncoefficients, in some embodiments of the invention. For example, apartition coefficient for a species can be compared to the partitioncoefficients for the species under different conditions, a partitioncoefficient for a species can be compared to the partition coefficientsfor the species when combined with other species, a set of partitioncoefficients for a species can be compared to other sets of partitioncoefficients, etc. The pattern obtained from a series of partitioncoefficients, e.g., vs. m/z using mass spectrometry or elution time forHPLC or other chromatographic techniques, etc., can be compared to otherpatterns obtained under different conditions, etc. In the case of massspectrometry analysis, the signal value at each m/z for, e.g., the topphase of a partitioning system may be divided by the signal value at thesame m/z from the bottom phase of the same partitioning system in somecases to yield a value of the partition coefficient at the same specificm/z. As another non-limiting example, the absorbance values at eachdesired time in an HPLC chromatogram for the top phase may be divided bytheir counterpart bottom values corresponding to the same time to yielda chromatogram of the partition coefficients, in some instances. Thesespectra or chromatograms may also be referred to as patterns or datapatterns in the present invention. This comparative information can beobtained, in some cases, at the same time or near the same time and inthe same system or a similar system as is used to determine theinteraction characteristics of the molecules of interest, or can beprovided as pre-prepared data in the form of charts, tables, orelectronically stored information (available on the Internet, disc,etc.).

In one embodiment of the present invention, proteins or otherbiomolecular mixtures from an experimental sample and from a referencesample (determined simultaneously, previously, or subsequently, asdescribed above) may be caused to partition in a variety of differentaqueous two-phase systems, e.g. formed by different types of polymers,such as Dextran and PEG or Dextran and Ficoll, by the same types ofpolymers with different molecular weights, such as Dextran-70 andPEG-600 or Dextran-70 and PEG-8,000, by the same polymers but containingdifferent in type and/or concentration salt additives, different buffersof different pH and concentration, etc. In some cases, the overallpartition coefficients for the mixtures determined using a particularassay procedure (same for both samples) can be determined in all of thesystems. In one embodiment, the systems displaying different partitioncoefficients for the mixtures under comparison may be selected as aseparation medium, for example, for further fractionation and/orcharacterization of the mixtures. In another embodiment, mixtures arepartitioned or otherwise separated using one or more standard systemswith known properties, e.g., those providing enhanced sensitivity levelstowards hydrophobic or ionic interactions. In such cases, the pattern ofindividual partition coefficients of the species comprising the mixturesmay be determined following separation of the mixtures in the phasesand/or compared between two or more mixtures.

The reasons for the observed differences in the partition behavior ofthe two samples do not necessarily have to be scientificallycharacterized for such differences to be useful for many applications,e.g., for diagnostics. Such differences, resulting in partitioningbehavior, may arise due to multiple reasons, including relativecompositional, structural, or conformational differences in the sampleswhen exposed to aqueous media of different solvent structures. Also, theidentity of the species contained in the pattern of partition behaviorof the two samples need not be known for the differences between the twopatterns to be useful for certain applications.

In one set of embodiments, the systems and methods proposed hereinprovide techniques for the separation and fractionation of proteinswhile preserving complexes and biomolecular interactions that may be ofinterest to distinguishing among samples. The solvent media in aqueouspartitioning may be selected to be compatible with the mixture ofbiomolecules. The solvent media may also be selected to preserve thehigher-order structures, as well as non-covalent binding amongbiomolecules such as proteins, small molecular weight ligands, etc. Forexample, appearance or disappearance of complexes by the methods of thisinvention can be useful for diagnostics and other applications. As aconsequence of such embodiments, the partition coefficients at anyspecific m/z or elution time may reflect the presence or absence of suchbiomolecular interactions.

One aspect of the present invention provides systems and methods able todistinguish among different samples, without being rigidly tied to fewseparation dimensions or variables, such as charge and/or size. Onenon-limiting example application of the present invention is to providean adjustable separation dimension, in which changes to the pattern ofindividual species can be uncovered via determination of their patternof individual partition coefficients or data patterns, enablingdetection and identification of changes that cannot be detected usingconventional separation means, such as molecular size or charge, and inwhich the absolute levels of concentration of such individual species isnot reflected in the pattern itself.

One embodiment of the present invention provides systems and methods fordiscovering a pattern of biomolecules in a biological sample, which, insome cases, may be changed between normal and diseased state of theunderlying organism. In some cases, a set of typically multiple systems,each known to provide sensitivity to structural changes leading todifferences in their hydrophobic, ionic, etc. interactions with theinteracting components, can be tested with the same samples. One or morepatterns can be identified as markers in one or more systems usingtechniques described herein, in certain embodiments. This marker ormarkers can also subsequently be used for diagnostics applications.

In yet another embodiment, a set of markers and the associated systemsin which such markers were discovered can be used for diagnosticsscreening. For example, the diagnostics test can include one or more ofthe following steps which can be carried out in any order suitable forsuch screening: (1) Partitioning or otherwise separating the sample inone or more of the systems which were used during the marker discoverystudy; (2) Processing the partitioned sample to obtain two or morepatterns of species concentration vs. m/z or elution time; (3) From eachtwo corresponding patterns, calculating the comparative pattern ofpartition coefficients vs. m/z or elution time; (4) Comparing thecomparative patterns to those representing normal and diseased stateswhich were obtained during the marker discovery study using anycombination of statistical or mathematical techniques; and (5) Denotinga diagnostics based on such a comparison, alone or in conjunction withother information.

As a specific non-limiting example, without loss of generality,comparing patterns of data obtained form at least two phases of apartitioning system, or from one phase and the original system, mayresult in at last two typical cases. In some cases, at the same (orpractically the same) ordinate parameter used to describe the pattern,e.g., a specific m/z value or elution time, two finite values of themeasured physical property (e.g., concentration) may be found in the twophases. In some cases, a partition coefficient specific to the ordinatelocation can be mathematically defined as the ratio of such properties.

In other cases, at the same (or practically the same) ordinate location,the sample from one of the phases may display a finite value of themeasured physical property while the other does not. Such a case maymean that the individual species corresponding to that ordinate locationwas totally or practically totally separated into one of the phases. Incertain instances, the partition coefficient specific to the ordinatelocation can be mathematically described as zero, infinity, or othercategorical rather than numerical values.

As a specific, non-limiting example of the first case, a pattern of themeasured property and the processed pattern of partition coefficientsmay be as follows: m/z 350 400 1100 12000 13500 17000 Top phase 30004000 1000 5000 14000 28000 peak Bottom phase 1000 2000 2000 7500 14000100000 peak Partition 3 2 0.5 0.714 1 0.28 Coefficient

As a specific, non-limiting example of the second case, a pattern of themeasured property and the processed pattern of partition coefficientsmay be as follows: m/z 100 450 1500 11000 11500 18000 Top phase 3000 0 05000 14000 0 peak Bottom phase 0 2000 2000 0 0 1000 peak Partition INFZERO ZERO INF INF ZERO CoefficientWithout loss of generality, these patterns could be described by thefollowing four cases, according to such embodiments:

1. A pattern of partition coefficients vs. an ordinate location.

2. A pattern of difference of the property values at specific ordinatelocation vs. the ordinate location.

3. A pattern of categorical values corresponding to zero or infinitypartition coefficient values vs. an ordinate location.

4. A mix of any of the above cases. In such a case, a pattern iscomprised of a series of numbers and categorical values vs. the ordinatevalue, together with a corresponding series of symbolic designators atthe same ordinate values that provides for annotation of the meaning ofthe specific entry in the pattern.

As another non-limiting example, a pattern may be described as: m/z 10004500 15000 19000 20000 21000 Partition Coefficient INF 0.35 ZERO 5.1 INF1.15

Without a loss of generality, searching for a biomarker or a set ofbiomarkers (e.g., to increase clinical specificity), according to someembodiments, and denoting a disease can involve one or more of thefollowing steps, carried out in any suitable order:

1. Preparing one or more aqueous two-phase partitioning systems.

2. Adding samples of plasma (homogenized tissue, urine, saliva, etc.)corresponding to normal and diseased state origins.

3. Partitioning the samples in the aqueous two-phase systems.

4. Removing aliquots from both phases of the aqueous two-phase systems(or from one phase and the original sample) for each sample. After thisstep there will be two aliquots for each sample.

5. Optionally, performing additional separation steps (e.g., HPLC orabsorbance to solid support favoring certain classes of proteins) toseparate groups of proteins in each aliquot according to a specificphysical property, e.g., size or charge.

6. Generating a mass spectra pattern of the sample in each aliquot usingmass spectrometer vs. its m/z value.

7. Calculating the ratio of the point-by-point corresponding spectraldata in each set of aliquots from the same sample, using categoricalvalues to denote absence of the property value at certain m/z value ineither of the two phases.

8. Using mathematical or statistical techniques, comparing thecomparative patterns of the ratios and/or categorical values for thenormal vs. diseased states.

9. Selecting one or more patterns, together with the partitioningsystems and other separation steps used to define such patterns, aspotential biomarker by their designation as different using themathematical or statistical techniques for the two types of samples.

It should be noted that discovering and selecting the marker(s), asdiscussed above, does not necessarily require the protein to beidentified. In some cases, the marker may comprise a pattern of speciesselected in the manner described herein. In one embodiment, the specificcomposition of the aqueous two-phase partitioning system or other systemmay be used to determine the ratio as being the partition coefficients.Multiple partitioning systems of different compositions can also be usedin methods similar to the ones described above. The selection of a setof markers for subsequent diagnostics may also depend on factors such asthe competing attributes of the increase in specificity, costs whenadditional biomarkers are included in the final set, or the like.

Once a set of biomarkers is discovered using techniques similar to thosedescribed above, a diagnostics screening test can be devised, accordingto some embodiments of the invention. As a non-limiting example, withoutloss of generality, a test may be conducted as follows:

1. Obtain a sample of plasma (homogenized tissue, urine, saliva, etc.)corresponding to unknown state (normal or diseased).

2. Add aliquots of the sample to the partitioning system used during thediscovery of the biomarkers. If more than one system was used, repeatthe same step for each different partitioning system.

3. Perform partitioning of the sample in each of the systems.

4. Perform any additional separation steps in accordance with the stepsused to define and select the patterns that correspond to the differentstates of the samples.

4. Obtain the mass spectrum of each of the partitioned phases.

5. Calculate the pattern of partition coefficients for each of thepartitioned phases (use categorical values as necessary as describedabove).

6. Compare, using appropriate mathematical or statistical techniques thecomparative pattern comprised of partition coefficients and/orcategorical values from the sample of unknown origin to thosecorresponding to the normal and diseased states.

7. Classify the unknown sample as diagnostically similar to one of theknown samples.

In some embodiments, the biomarker may represent a mixture of forms ofthe same protein, and/or mixtures which complex between biomolecules orbetween biomolecules and other molecules that may appear or disappearbetween normal and diseased states. Changes in the distribution orrelative amounts of the different forms of the same protein may result,in some cases, in a different partitioning behavior of the same protein,and appearance or disappearance of complexes may result in theappearance or disappearance of, e.g., spectral peaks.

Some aspects of the invention provide a variety of studies, at the levelof determining tools for physiological analysis and/or for carrying outphysiological analysis. For example, tools for determining analysisprocedures can involve taking samples from a single individual ormultiple individuals. In one embodiment, a positive sample and a controlsample can be taken from a single individual. For example, an individualmay have a tumor and a positive sample may be a portion of the tumor,where a control sample is from a non-tumorous portion of the individual.The samples, both positive and control, can be taken from the individualat the same time or at different times. For example, samples from atumorous portion of an organism can be taken at different times, andused to determine differences in the patterns of the samples as toolsfor analysis of the progression of a tumor.

In some cases, single patterns or multiple patterns can be used asmarkers. Multiple patterns from a single sample can be identified asseparate markers for a particular condition, and during analysis,separate patterns can be studied in certain instances. As one example, asingle pattern can define a marker identified by and/or studied inconnection with a single partitioning system. In another embodiment,multiple patterns from a single sample can be identified as separatemarkers for a particular condition using multiple partitioning systems,and during analysis separate patterns can be studied.

The analytical tool used to evaluate the pattern of partitioncoefficients or the categorical values may be, e.g., a massspectrometer, liquid chromatography such as HPLC, or other spectraltechniques such as UV, IR, Raman or other absorbance and/or scatteringtechniques. The ordinate for the pattern in each case depends on thetechnique, for example, m/z for mass spectra, time for HPLC, wavelengthfor most spectral techniques, etc. The technical aspects of the methodmay result in a pattern of one or more peaks (e.g., values at specificm/z) or a diffuse pattern obtained as a summation of responses from manymolecules at each specific wavelength (e.g., an HPLC chromatogram orUV-Vis absorbance spectrum). The mathematical techniques used to analyzesuch patterns may vary depending on the technique used, as is understoodby those of ordinary skill in the art, and the relative choice of eachanalytical tool will be determined primarily by its sensitivity,resolution, and other operational characteristics without a loss ofgenerality of the present invention.

Mathematical and statistical pattern recognition techniques may be used,in some cases, to analyze the data, including linear and non-lineartechniques, such as principal component analysis, partial least squares,artificial neural networks, genetic algorithms, Fourier or wavelettransforms, etc. One or more such algorithms may be used to process,transform, condense, or manipulate the series of partition coefficientand/or categorical values, in some cases. The raw values or theirprocessed data corresponding to a given mixture of species (e.g., serumsample from a positive case) may be compared using such techniques tosimilarly obtained and processed data corresponding to a differentmixture of species (e.g., from a negative case). During patterndiscovery, such techniques may be provided with multiple examples ofsamples of different classes and analytical discriminatory aspects ofthe data are discovered and presented in mathematical or statisticalmanner. In certain instances, such techniques may make use thediscriminatory aspects previously discovered to interrogate new dataobtained from similarly conducted experiments and subsequentlycritically compare such data to previously known cases foridentification.

According to one aspect of the present invention, a computer and/or anautomated system is provided able to automatically and/or repetitivelyperform any of the methods described herein. As used herein, “automated”devices refer to devices that are able to operate without humandirection, i.e., an automated device can perform a function during aperiod of time after any human has finished taking any action to promotethe function, e.g. by entering instructions into a computer. Typically,automated equipment can perform repetitive functions after this point intime. One specific non-limiting example of a technique that can make useof a computer or other automated system is in a process in which aphysiological condition of a system as determined by determining arelative measure of interaction between one or more species from asample from the system and various interacting components of apartitioning system. In the clinical setting, this may be accomplished,for instance, by drawing a sample of blood (milliliter-sized or a verysmall sample such as a drop or less) and subjecting the blood sample ora subset thereof (e.g., plasma) to a multi-phase partitioning process.The results of this process can then be compared to similar behavior ofmarkers in a similar system, which can take the form of data storedelectronically.

FIG. 1 is a schematic block diagram of an example system according toone embodiment of the present invention. In the embodiment illustratedin FIG. 1, a controller 200 is implemented on a conventional personalcomputer 250 that includes a processor 251, a memory 252, an inputdevice 253, optionally a removable storage device 254, a pointing device255, a display device 256, and a communication device 257, all coupledtogether via a bus 258. In a conventional manner, memory 252 may includea variety of memory devices, such as hard disk drives or optical diskdrives, RAM, ROM, or other memory devices and combinations thereof, andinput device 253 may include a keyboard, a microphone, or any other formof input device capable of receiving one or more inputs 210 from a userof controller 200. Removable storage device 254 may include a CD-ROMdrive, a tape drive, a diskette drive, etc. and may be used to loadapplication software, including software to implement variousembodiments of the present invention described herein. Display 256 mayinclude a conventional CRT display screen, a flat panel display screen,or any other type of display device that allows textual information tobe displayed to the user, and pointing device 255 may include a puck, ajoystick, a trackball, a mouse, or any other type of pointing device orscrolling device that permits the user to select from among the varioustextual information displayed on the display device 256. Communicationdevice 257 may include any form of communication transceiver capable ofreceiving one or more inputs 220 from the fluid-handling apparatus 30and providing one or more outputs to the fluid-handling apparatus 30.For example, communication device 257 may include a RS232/485communication transceiver, a 4-20 mA analog transceiver, an Ethernettransceiver, etc.

Software, including code that implements embodiments of the presentinvention, may be stored on some type of removable storage media such asa CD-ROM, tape, or diskette, or other computer readable mediumappropriate for the implemented memory 252 and the removable storagedevice 254. The software can be copied to a permanent form of storagemedia on the computer 250 (e.g., a hard disk) to preserve the removablestorage media for back-up purposes. It should be appreciated that inuse, the software is generally and at least partially stored in RAM, andis executed on the processor 251.

Various embodiments of the present invention can also be implementedexclusively in hardware, or in a combination of software and hardware.For example, in one embodiment, rather than a conventional personalcomputer, a Programmable Logic Controller (PLC) is used. As known tothose skilled in the art, PLCs are frequently used in a variety ofprocess control applications where the expense of a general purposecomputer is unnecessary. PLCs may be configured in a known manner toexecute one or a variety of control programs, and are capable ofreceiving inputs from a user or another device and/or providing outputsto a user or another device, in a manner similar to that of a personalcomputer. Accordingly, although embodiments of the present invention aredescribed in terms of a general purpose computer, it should beappreciated that the use of a general purpose computer is exemplaryonly, as other configurations may be used.

As shown in FIG. 1, the controller 200 is adapted to be coupled to afluid handling apparatus 30, to control operation of the fluid handlingapparatus. Controller 200 includes an input 210 to receive one or moreparameters from a user of the controller 200 relating to the desiredoperation to be performed. The controller 200 also includes a pluralityof inputs 220 to receive signals relating to the operational status ofthe fluid handling apparatus, and a plurality of outputs 230, 240 toconfigure and control the fluid handling apparatus. User inputparameters received on input 210 may include the type and amount ofprotein and/or other biomolecules that is to be processed by the fluidhandling apparatus, the compositions of liquids used by the fluidhandling apparatus for, e.g., liquid-liquid partitioning, etc.

Some embodiments of the present invention permit the user to specify oneor a number of input parameters relating to the operation of the fluidhandling apparatus, and then, based upon the input parameters, toconfigure and control the fluid handling apparatus. Depending upon thenumber of input parameters specified by the user, the controller mayprompt the user for additional parameters prior to configuring the fluidhandling apparatus.

Inputs 220 of controller 200 are adapted to receive a plurality ofsignals relating to the operational status of the fluid handlingapparatus. Signals that may be received on inputs 220 generallycorrespond to physical conditions within the fluid handling apparatus,and may include, for example, the concentration of proteins or othermolecules within the fluid handling apparatus, the time of exposure, thetime for settling to occur, the degree of agitation, the operatingtemperature or pressure, etc.

Outputs 230, 240 of the controller 200 are adapted to configure andcontrol the fluid handling apparatus, based upon the user parametersreceived at input 210, and optionally, one or more of the signalsreceived on inputs 220. Output 230 may provide a number of separatesignals, for example, a signal to introduce a protein or other moleculewithin a liquid, a signal to control the operating temperature, etc.

According to another embodiment of the present invention, controller 200may include a database and/or a knowledgebase that can be accessed byprocessor 251. According to one embodiment of the present invention, thedatabase may include a plurality of records, each record correspondingto a particular set of parameters for which the fluid processingapparatus may be used to determine a relative measure of interaction.Unless specifically indicated otherwise, as used hereinafter, the term“parameters” is used to refer to both process parameters (e.g., theamount of protein or other biomolecule(s) to be added, the operatingtemperature etc.), as well as characteristics (e.g., concentration,separation time, etc.) of the experiment given a particular set ofprocess parameters. In general, each of the records stored in thedatabase reflects empirical data based upon use of the fluid processingapparatus under defined conditions, or the use of a similar fluidprocessing apparatus under defined conditions. The controller 200 andthe database may thus be viewed as forming an “expert” system. Thedatabase may be stored on a removable storage medium and copied tomemory 252 for use by the processor 251, or alternatively, thecontroller may be pre-configured to include the database.

In some cases, the database (or knowledgebase) may be configured for aparticular type of fluid handling apparatus (e.g., a specific model froma particular manufacturer of fluid handling apparatus), or may beconfigured to be used with a variety of types of fluid handlingapparatuses. In some cases, the database may be configured for aparticular type of protein and/or other biomolecule. Alternatively, amore general database may be used that includes a number of differentproteins, biomolecules, aqueous solutions, etc. with which a variety ofdifferent fluid handling apparatuses may be used. In use, the databasemay be accessed by a fluid handling apparatus configuration and controlroutine that is performed by controller 200 to configure and controlfluid handling apparatus 30 that is operatively coupled thereto. Itshould be appreciated that while the database or knowledgebase isinitially based on empirical data obtained with similar equipment, thedatabase may be periodically updated (e.g., new records may be addedand/or existing records may be modified) to reflect additional dataobtained in use, or by use of similar equipment. Another aspect of thedatabase is related to its capacity for storage and retrieval of patterninformation related to raw or processed data from the analysisinstrumentation. Such data might include sequence of values, categoricalinformation, mathematical coefficients, and other method-specific andsample-specific information useful for discovery and use of suchpatterns for the applications described herein.

The techniques and apparatus described herein can be used to discovermarkers or to execute a diagnostics test. The apparatus could beinterfaced to other devices and instruments known to those skilled inthe art, including automated sample preparation instruments, liquidchromatography columns, HPLC systems, mass spectrometers, absorbanceinstruments, etc. Data obtained from such devices and instruments couldbe electronically channeled to a software for performing data reductionand analysis and for delineating a diagnostics.

The following examples illustrate the analysis of patterns obtained fromdifferent experimental data for diagnosis applications. These examplesare intended to illustrate certain embodiments of the present invention,but not exemplify the full scope of the invention.

EXAMPLE 1 Using 2D-HPLC Data to Discover Patterns in Elution Profilesthat, when Considered Together with their Diagnosis, may Provide Meansto Determine the Latter in Unknown Samples

This example is provided to illustrate the use of an embodiment of theinvention for diagnosis purposes, and describes one technique providedby the present invention and a methodology for analyzing a medicalcondition, but is not intended to provide a specific marker oridentifier for a specific medical condition.

Blood samples containing 4.5 mL were drawn from healthy donors (control)and patients with post-traumatic stress disorder (PTSD). The bloodsamples were collected into glass BD Vacutainer tubes containing 0.5 mL3.2% sodium citrate and centrifuged at 1,000 rpm for 45 min at 8° C. Theplasma was carefully removed, aliquoted and frozen at −80° C. Thesamples were thawed and subjected to partitioning in a aqueouspoly(ethylene glycol)-sodium sulfate-Na/K-phosphate buffer, pH 7.4two-phase system.

The aqueous two-phase system used in these experiments contained 15.70wt.% polyethylene glycol-600 (with a molecular weight of about 600),9.47 wt.% sodium sulfate, and 2.30 wt.% Na/K-phosphate buffer, pH 7.4.The systems were prepared by mixing the appropriate amounts of stockpolymer, salt, and buffer solutions by weight into a 100×75 mm tube upto a total weight of a system of 4.00 g. The ratio between the volumesof the two phases of each system (volume of the top phase to volume ofthe bottom phase) was about 1:1. A fixed amount of 600 microliters ofblood plasma was added to a system. The system was vigorously shaken andcentrifuged for 30 min at about 3500 rpm in a centrifuge with a bucketrotor to speed the phase settling. The tubes were taken out of thecentrifuge, and samples from the top and the bottom phases werewithdrawn. Aliquots containing about 0.3 ml from the top phase and 1.7ml from the bottom phase were withdrawn, and each aliquot was dilutedwith starting buffer (Beckman-Coulter, Fullerton, Calif., USA) to 2.50mL total volume. Each sample was vortexed and subjected to bufferexchange using PD-10 column (Amersham Pharmacia Biotech) as follows. ThePD-10 column was equilibrated with 25 mL of start buffer. Each sample(2.5 mL) was loaded onto a PD-10 column, the column was washed withstart buffer, and the first 2.5 mL fraction was collected.

A ProteomeLab PF 2D system from Beckman-Coulter (Fullerton, Calif.) wasused for the 2D-HPLC analysis. Following the above procedure, a sampleof 2.00 mL was injected, and first dimension separation was performedusing a standard procedure with a flow rate of 0.2 mL/min, and bymonitoring of the absorbance of the column effluent at 280 nm. Duringthe pH gradient portion of the run extending from 8.0 to 4.0 pH,fractions at 0.3-pH intervals were collected as detected by a pHmonitor, which controlled the fraction collector. Each collectedfraction was subjected to second dimension separation by Reverse-PhaseHPLC (RP-HPLC) using standard protocols from the manufacturer. TheRP-HPLC experiments using 200 microliters volume of each fractioninjected was performed at 50° C. with a flow rate of 0.75 mL/min, andabsorbance of the column effluent was monitored at 214 nm.

Chromatograms obtained under second dimension RP-HPLC for thecorresponding plasma fractions (collected for the same pH intervals)from the samples obtained from the top and bottom phases were digitallystored on a system computer. Chromatograms of samples from healthydonors and from PTSD patients were compared after the second separationdimension at fixed pH intervals. In each case, a comparative spectrum(herein, a comparative chromatogram) of the point-by-point partitioncoefficients was constructed by dividing the data obtained from the topand the bottom of each sample. Additional mathematical operations toshift, smooth, clip, or transform any data pattern are totallyarbitrary, as long as they are performed in the same manner on eachsample. The comparative spectrum originating from a healthy donor(control) is shown in FIG. 3, and the corresponding comparative spectrumfrom a PTSD patient is shown in FIG. 4, both obtained from fractionshaving a pH range of 6.0 and 6.4. The two comparative spectra arevisually different. Further mathematical and statistical techniques tocompare the degree of similarity between the two spectra could readilybe performed using automated procedures to classify additional samplesof unknown diagnosis as similar to either of the samples analyzedherein. Such techniques could be used to arrive at a diagnosis usingsuch a comparison, or more typically, in conjunction with other data andinformation. It should be noted that in some applications, many samplesbelonging to both negative and positive states of a diagnosis will beprocessed and combined by techniques known to those skilled in the artto define diagnosis tools that are statistically valid with respect tosensitivity and specificity levels.

EXAMPLE 2 Patterns of Mass Spectra Could be Used to Analyze SerumSamples Obtained from Healthy and Ovarian Cancer Patients

This example is provided to illustrate the use of an embodiment of theinvention for diagnosis purposes, and describes one technique providedby the present invention and a methodology for analyzing a medicalcondition, but is not intended to provide a specific marker oridentifier for a specific medical condition.

Pooled serum samples from healthy (sample identifier 0651) and ovariancancer patients (sample identifier 4850) were obtained from the ClinicalProteomics Reference Laboratory (Gaithersburgh, Md.). The aqueoustwo-phase system used in these experiments contained 15.70 wt. %polyethylene glycol-600 (with a molecular weight of about 600), 9.5 wt.% sodium sulfate, 4.8 wt. % NaCl, and 0.64 wt. % Na/K-phosphate buffer,pH 7.4. Several such systems were prepared by mixing the appropriateamounts of stock polymer, salt, and buffer solutions by weight intomicrotubes up to a total weight of each system of 0.425 g. The ratiobetween the volumes of the two phases of each system (volume of the topphase to volume of the bottom phase) was about 1:1. A fixed amount of 75microliters of blood serum was added to a system. The system wasvigorously shaken and centrifuged for 30 min at about 3500 rpm in acentrifuge with a bucket rotor to speed the phase settling. Themicrotubes were taken out of the centrifuge, and samples from the topand the bottom phases were withdrawn. Aliquots of 60 microliters fromthe top phase and 60 microliters from the bottom phase were withdrawnand dispensed into separate microtubes followed by addition of 240microliters of water into each microtube.

The aliquots were sent for mass spectra analysis at the ClinicalProteomics Reference Laboratory using Surface Enhanced Laser DesorptionIonization mass spectrometer (Ciphergen, Fremont, Calif.) according tothe following protocol: (1) a Q10 chip (Ciphergen) was twice treatedwith phosphate buffer for 5 minutes; (2) each aliquot was added to eachwell of the chip and incubated for 60 minutes at room temperature; (3)the chip was washed three times with 150 microliters of phosphate bufferusing 10 mixing cycles, followed by a single wash with water; (4) Thechip was air dried for 10 minutes; (5) 1 microliter of SPA matrix in 50%acetonitrile/water with 0.5% TFA was added to the chip, which was thenair dried for 15 minutes; (6) step (5) was repeated. The chip was thenplaced into the mass spectrometer. The above protocol was repeated oncefor each sample (two repeats in total).

Raw spectral intensity data and the total ion current for each samplewere sent back to ANALIZA, Inc. (Cleveland, Ohio) for further analysis.Each spectral data vector, having pairs of mass over charge (m/z) andintensity values, was normalized with respect to its total ion current,then averaged with a second vector corresponding to the second repeat ofthe same sample. The averaged spectral data vectors of the top andbottom aqueous phases corresponding to the same sample were divided on apoint-by-point m/z basis, resulting in a data vector of the relativemeasure of interaction, K, versus m/z. This protocol was repeated forboth healthy and cancer pool samples.

A comparison of the spectral K vector for the two samples is shown inFIG. 5, for a selected range of m/z values. Consistent differences overa range of m/z between the cancer and the normal pooled samples mayindicate a potential biomarker for early screening and/or otherdiagnostics applications. Patterns of the relative measure ofinteraction vs. m/z can be developed from such data and subsequentlyused for diagnostics applications using techniques known in the art.Specific biomarkers could also be identified using mass spectrometry andother techniques and subsequently used to develop direct assays formeasuring the relative measure of interaction specific to a biomarkerusing immunoassay and other techniques known in the art.

Biomarkers developed using techniques described in the present inventioncan also be used to distinguish between early and later stage ovariancancer, as illustrated in FIG. 6. The experimental protocol used wasidentical to the protocol described herein in Example 2. The two normalsamples included different pools and exhibited certain variability.However, the variability between the two normal samples is significantlyless than that between the early and late cancer samples. Other uses ofbiomarkers described in the present inventions could be developed fordifferent applications.

EXAMPLE 3 Patterns of Mass Spectra Discovered Using Present Inventioncould have Certain Advantages Over Expression-Based Spectra

This example is provided to illustrate the use of an embodiment of theinvention for diagnosis purposes, and describes one technique providedby the present invention and a methodology for analyzing a medicalcondition, but is not intended to provide a specific marker oridentifier for a specific medical condition. This example furtherillustrate a certain advantage of biomarkers that are discovered usingtechniques of the present invention over biomarkers that are discoveredusing conventional protein expression proteomics techniques.

Experimental techniques used in the present example are more fullydescribed in Example 2, but with an aqueous two-phase system ofdifferent composition. An aqueous two-phase system contained 18.0 wt. %Ficoll-70 (with a molecular weight of about 70,000), 13.0 wt. %Dextran-75 (with molecular weight of about 75,000), 0.15 M NaCl, and0.01 M Na/K-phosphate buffer, pH 7.4. Normalized differences defined as100 (normal value−cancer value)/normal value were calculated from thedata for the relative measure of interaction as described herein for thetotal protein expression. The data in FIG. 7 illustrates that thenormalized differences between normal and cancer for the relativemeasure of interaction were significantly more distinct than thoseobtained using protein expression at the same m/z values. Thisobservation is important for practical reasons, since it is wellrecognized that the natural variability of expression, which is notrelated to the underlying disease process, is a major hindrance in thediscovery and the clinical use of expression level biomarkers, includingm/z patterns of the same. In practice, differences of 50% in expressionlevels are sometime well within the natural variability bound andexpression patterns as illustrated in FIG. 7. The same samples, whenanalyzed using the relative measure of interaction and its m/z pattern,have resulted in significantly more distinct differences between normaland cancer samples, which could be used to delineate the clinical originof an unknown sample by its similarity to the pattern shown herein.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of”, when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

1. A method of determining at least one characteristic of a plurality ofspecies, comprising: exposing a plurality of species to an aqueouspartitioning system including at least first and second phases;obtaining, using mass spectroscopy, a first spectral data patterncomprising cumulative spectral information from a first sample of one ormore species associated with the first phase of the aqueous partitioningsystem; obtaining, using mass spectroscopy, a second spectral datapattern from one or more of the following: (1) a second sample of one ormore species associated with the second phase of the aqueouspartitioning system, or (2) a portion of the plurality of species priorto the exposing step; and comparing at least a portion of the firstspectral data pattern with at least a portion of the second spectraldata pattern to determine at least one characteristic of a plurality ofspecies.
 2. The method of claim 1, comprising performing the methodwithout determining the identity of at least one of the species in theplurality of species after exposing the at least one species to theaqueous partitioning system.
 3. The method of claim 1, comprisingperforming the method without determining the level, abundance,concentration, and/or identity of any of the plurality of species afterexposing the plurality of species to the aqueous partitioning system. 4.The method of claim 1, comprising performing the method without usinginformation related to the level, abundance, concentration, and/oridentity of any of the plurality of species after exposing the pluralityof species to the aqueous partitioning system.
 5. The method of claim 1,comprising performing the method without determining the identity of atleast one of the species in the plurality of species prior to exposingthe at least one species to the aqueous partitioning system.
 6. Themethod of claim 1, comprising performing the method without determiningthe level, abundance, concentration, and/or identity of any of theplurality of species prior to exposing the plurality of species to theaqueous partitioning system.
 7. The method of claim 1, comprisingperforming the method without using information related to the level,abundance, concentration, and/or identity of any of the plurality ofspecies prior to exposing the aqueous partitioning system.
 8. The methodof claim 1, comprising performing the method without determining theidentity of at least one of the species in the plurality of speciesprior to or after exposing the at least one species to the aqueouspartitioning system.
 9. The method of claim 1, comprising performing themethod without determining the level, abundance, concentration, and/oridentity of any of the plurality of species prior to or after exposingthe plurality of species to the aqueous partitioning system.
 10. Themethod of claim 1, comprising performing the method without usinginformation related to the level, abundance, concentration, and/oridentity of any of the plurality of species prior to or after exposingthe plurality of species to the aqueous partitioning system. 11.(canceled)
 12. The method of claim 1, comprising: comparing the firstspectral data pattern and the second spectral data pattern to define acomparative pattern; and comparing the comparative pattern to areference pattern to determine the at least one characteristic. 13.(canceled)
 14. The method of claim 1, wherein the plurality of speciesis first fractionated prior to the act of exposing the plurality ofspecies to the aqueous partitioning system. 15-17. (canceled)
 18. Themethod of claim 1, wherein the first and second data patterns arecompared without determining partition coefficients of a species of theplurality of species between the first and second phases. 19-20.(canceled)
 21. The method of claim 1, wherein the plurality of speciespartitions between the first and second phases on the basis ofstructural differences.
 22. The method of claim 1, wherein the act ofcomparing comprises comparing at least a portion of the first spectraldata pattern with at least a portion of the second spectral data patternto determine a condition of a biological system.
 23. (canceled)
 24. Themethod of claim 1, wherein the second data pattern is representative ofa sample indicative of an unknown condition of an organism, and thesecond data pattern is derived from samples indicative of both normaland abnormal conditions of the organism. 25-29. (canceled)
 30. Themethod of claim 1, wherein the method is performed without determiningthe chemical or biological identity of any of the plurality of species.31-38. (canceled)
 39. A method of determining at least onecharacteristic of a plurality of species, comprising: exposing aplurality of species to at least first and second interacting componentsto at least partially separate the plurality of species; treating afirst sample of the at least partially separated plurality of species,using mass spectroscopy, to produce a first spectral data pattern;treating one or more of the following, using mass spectroscopy, toproduce a second spectral data pattern: (1) a second sample of the atleast partially separated plurality of species that is not identical tothe first sample, or (2) a portion of the plurality of species prior tothe exposing step; and comparing at least a portion of the firstspectral data pattern with at least a portion of the second spectraldata pattern to determine at least one characteristic of a plurality ofspecies. 40-48. (canceled)
 49. A method of determining at least onecharacteristic of a plurality of species, comprising: exposing aplurality of species to an aqueous partitioning system including atleast first and second phases; obtaining a first data pattern comprisingcumulative information from a first sample of one or more speciesassociated with the first phase of the aqueous partitioning system;obtaining a second data pattern comprising cumulative information bytreating one or more of the following: (1) a second sample of one ormore species associated with the second phase of the aqueouspartitioning system, or (2) a portion of the plurality of species priorto the exposing step; and comparing at least a portion of the first datapattern with at least a portion of the second data pattern to determineat least one characteristic of a plurality of species. 50-56. (canceled)57. A method of determining at least one characteristic of a pluralityof species, comprising: exposing a plurality of species to at leastfirst and second interacting components defining at least a first phaseand a second phase, respectively, of a first system that includes atleast two phases; obtaining a first spectral data pattern comprisingcumulative spectral information from a plurality of the speciesassociated with the first phase of the system after exposure; obtaininga second spectral data pattern comprising: cumulative spectralinformation from a plurality of the species associated with the secondphase of the system after exposure, and/or cumulative spectralinformation from a plurality of the species prior to exposure to thesystem; and deriving comparative spectral information from comparison ofat least a portion of the first spectral data pattern with at least aportion of the second spectral data pattern, to determine acharacteristic of a plurality of species. 58-102. (canceled)